1. Field of the Invention
This invention relates to estrogen receptors and ligands for them, and in particular to crystalline estrogen .beta. receptor (ER.beta.) and to methods of identifying ligands utilizing crystalline ER.beta..
2. Brief Description of the Art
The thyroid hormone receptor (TR) is known and its three-dimensional structure, and hence its ligand binding domain, has been determined. Knowledge of the three-dimensional structure has enabled a better understanding of the modes of ligand binding and the determination of the optimum conformation of ligand to bind to the receptor. It is generally believed in the art that the TR structure also provides a guide to the design of ER ligands.
Estrogen steroid hormone and thus the estrogen receptor (ER) is a member of the steroid hormone receptor family. Its primary natural ligand is estradiol (E2). However, it is known that a large number of structurally diverse non-steroidal compounds such as raloxifene, centchroman, coumestrol, diethylstilbesterol, esculin, tamoxifen, zearalenone, and zindoxifen also bind to the estrogen receptor (FIG. 4). The majority of these non-steroidal estrogen receptor ligands contain 2-4 carboxylic, aromatic, and/or heterocyclic rings connected by a 1-3 atom chain. One or more of the rings may be fused with the central atom chain or with each other.
It has been proposed that the receptor possesses a multi-functional modular structure potentially having discrete domains for DNA binding, ligand binding, and transactivation. The ligand binding domain (LBD) has been designated domain E and is the largest domain of the estrogen receptor. The ligand binding domain includes a ligand recognition site and regions for receptor dimerization interaction with heat shock proteins, nuclear localization and ligand dependent transactivation.
A review of the structure and functioning of the estrogen receptor is provided in an article by Katzenellenbogen, J. et al., Steroids, (1997) 62(3):268-303.
It is known that compounds which bind to the estrogen receptor are potentially useful in the treatment of a wide range of disease states. These include estrogen agonists for treatment of disease linked to estrogen deficiency (e.g. osteoporosis, cardiovascular and neurodegenerative diseases in post menopausal women) and estrogen antagonists for treatment of breast and uterine cancer. Furthermore, it is known that certain ligands such as tamoxifen display mixed agonist/antagonist action (that is they are either estrogen agonists, estrogen antagonists, or a partial estrogen antagonists when binding to the estrogen receptors of different tissues) and such compounds may simultaneously prevent bone loss and reduce the risk of breast cancer. It is further known that benzothiophenes are usable as agonists or antagonists to steroid hormones, and that it is possible to modify their binding mechanics, for example the binding affinity, by changing the substituent groups at various positions on the molecule. Therefore, it would be desirable to be able to design ligands which are recognizable by and able to bind to the estrogen receptor. Additionally, it would be desirable to know the three dimensional structure of the estrogen receptor. Such knowledge would be useful for the design of compounds intended to bind to the estrogen receptor. The present inventors have been able to produce an estrogen receptor crystal and to determine from that the three dimensional structure of the estrogen receptor. Unexpectedly, the thus determined ER structure reveals that the TR structure does not provide a good model for binding of ligands to ER.
Our copending patent application No. PCT/GB98/01708 discloses inter alia, the crystal co-ordinates of crystalline estrogen receptor alpha (ER.alpha.).